|Publication number||US4613566 A|
|Application number||US 06/573,013|
|Publication date||Sep 23, 1986|
|Filing date||Jan 23, 1984|
|Priority date||Jan 23, 1984|
|Publication number||06573013, 573013, US 4613566 A, US 4613566A, US-A-4613566, US4613566 A, US4613566A|
|Original Assignee||President And Fellows Of Harvard College|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (5), Non-Patent Citations (10), Referenced by (71), Classifications (13), Legal Events (6)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The invention described herein was made with Federal Government support and the Government has certain rights in the invention.
This invention relates to an improved assay method utilizing DNA hybridization from porous carrier sheets and pertains more specifically to an improved assay method for enabling simple cross-hybridization of a labelled probe with multiple test specimens.
The Southern blot-hybridization procedure (Southern, J.Mol.Biol., Vol. 98, 503-517 (1975)) has been widely employed for detecting specific sequences among DNA fragments separated by gel electrophoresis. The procedure has been modified and extended to a so-called "two-dimensional" matrix of hybridization spots as described by Sato et al., PNAS, Vol. 74, 542-546 (1977); and by Wensink et al., Cell, Vol. 18, 1231-46 (1979). The modified procedure involves transferring a pattern of electrophoresed spaced bands of a DNA digest, each band containing separate DNA fragments from the electrophoresis gel to a porous carrier sheet and bonding or immobilizing it thereon by baking, then placing in face-to-face contact with a second gel containing a pattern of electrophoresed bands of a labelled DNA digest with the gel bands rotated approximately 90° with respect to the bands in the carrier sheet, then causing the labelled DNA fragments in the gel to flow through the carrier sheet, thus giving the fragments an opportunity to hybridize with the immobilized carrier sheet bands at the spots where the bands cross. Determining the location of labelled spots in the carrier sheet, for example by autoradiography or fluorography, makes it possible to determine which of the DNA fragments exhibit nucleotide sequences complementary to each band of the other pattern. This procedure makes it possible to determine in a single assay the complementarity of the DNA fragment in each gel band with that of each band in the carrier sheet.
However, the assay procedure is inefficient in that hybridization can occur only during the very brief time that any particular labelled fragment is actually traversing the carrier sheet containing the unlabelled DNA fragment. The inefficiency of this step requires that the radiolabelled DNA have a high specific activity, usually requiring that the DNA be nick-translated. This in turn leads to degradation of the DNA and consequent loss of resolution during gel electrophoresis and an additional loss of resolution resulting from flow of the hybridization buffer during the hybridization step.
The present invention provides a hybridization assay of such greatly improved resolution and sensitivity as to make possible simultaneous comparison of all fragments from as many as 10 different test specimens of DNA or RNA with all specimens of a DNA or RNA probe. The invention also enables the use of end-labelled DNA fragments when radiolabels are used, thus decreasing or eliminating degradation of the DNA caused by nick translation. The invention also features providing both the probe and test fragments of DNA or RNA in separate porous carrier sheets saturated with hybridization buffer and maintained in contact with each other while subjected to hybridization conditions. The invention also features maintaining the hybridization buffer in static condition during the hybridization step.
The invention is the method of assaying by hybridization a plurality of different DNA test fragments which comprises providing a probe sheet in the form of a pattern of parallel bands of labelled material comprising DNA or RNA fragments on a porous carrier sheet saturated with buffer free from solvent capable of eluting RNA or DNA from the probe sheet, providing a first test sheet comprising a pattern of parallel bands of test material comprising DNA or RNA fragments, unlabelled or bearing a label different from the probe fragments, immobilized on a porous carrier sheet saturated with hybridization buffer free from solvent capable of eluting RNA or DNA from the probe sheet, placing said first test sheet in face to face contact with said probe sheet with said probe and test bands crossing each other, providing at least one additional test sheet comprising a pattern of parallel bands of test material comprising DNA or RNA fragments, unlabelled or bearing a label different from the probe fragments, immobilized on a porous carrier sheet saturated with hybridization buffer containing solvent capable of eluting RNA or DNA from the probe sheet, at least one of said materials being DNA fragments, stacking said additional test sheets in face to face contact with said first test sheet at the side opposite said probe sheet with their bands aligned with the bands of said first test sheet to form an assemblage comprising said probe sheet and all said test sheets, subjecting said assemblage to hybridizing conditions while maintaining said hybridization buffer in static condition, separating said test sheets from said assemblage and washing to remove unhybridized labelled material therefrom, and determining on each test sheet the location of labelled material hybridized to said test material.
In a preferred embodiment, only the probe DNA or RNA fragments are labelled, for example radiolabelled, and the test material remains unlabelled. However, it is possible to practice the invention with labelled test material as well, provided the label of the test material is different from the label of the probe material, hence distinguishable from it, as in the case of tritium and 32 P labelling. It will also be appreciated that the invention can be practiced by providing simultaneously a second stack of test sheets on the opposite side of the probe sheet from the first stack. This procedure speeds up the procedure since the probe materials need diffuse only half as far to penetrate the same total number of test sheets as when the test sheets are all stacked on one side of the probe sheet.
The labelling employed may be radiolabelling, for example as pointed out above, or may be other forms of labelling embodying phosphorescence, chemiluminescence, biotin-avidin complexing, and the like, as is well known in the art.
While the parallel bands of DNA or RNA fragments, labelled or unlabelled, may be formed by any procedure, the invention is particularly applicable to assaying a pattern of parallel bands formed by gel electrophoresis of a DNA restriction digest or of RNA, the pattern being transferred from the gel to a porous carrier sheet by the Southern blot or by other conventional procedure such as electrophoresis. Any conventional porous carrier sheet for the DNA or RNA fragments may be employed in the practice of the present invention; among the materials adsorptive of nucleic acids which can be used are nitrocellulose paper, diazobenzyloxymethyl paper, and neutral or positively charged porous adsorptive nylon, i.e. any solid film-forming polyamide having an affinity for biological molecules. Such porous membranes are described in U.S. patents Lovell et al U.S. Pat. No. 2,783,894 and Marinaccio et al. U.S. Pat. No. 3,876,738. Porous carrier sheets of 66-nylon (copolymer of hexamethylene diamine and adipic acid) are available from several sources including AMF Inc., White Plains, N.Y., Pall Corp., Glen Cove, N.Y. Porous carrier sheets are also available from New England Nuclear Corp., Boston, Mass. under the trade names "GeneScreen" (neutral) and "GeneScreen Plus" (positively charged). The "GeneScreen Plus" porous carrier sheet can be used as the porous carrier sheet for the test sheets, but not for the probe sheet.
In the case of the probe sheet containing the bands of labelled probe material, the material is simply adsorbed on or in the sheet by transfer from the electrophoresis gel in which it is formed, without immobilization or bonding of the fragments to the carrier sheet but instead leaving the labelled material free to diffuse into contact with the bands of unlabelled material in the test sheets during the hybridization step. The test material in the test carrier sheets, on the other hand, is immobilized or bonded to the sheets by any conventional procedure such as baking at a temperature of 50°-100° C. In the case of "GeneScreen Plus" no baking is required, contact of the test material with the carrier sheet at room temperature sufficing to cause bonding; consequently this carrier sheet cannot be used for the probe sheet.
Physical movement or flow of the transfer or hybridization buffer during the hybridization step should be minimized or eliminated in order to provide high resolution and sensitivity of the assay; that is, the assemblage of probe sheet and test sheets is subjected to hybridizing conditions while maintaining the buffer in static condition. This can most conveniently be accomplished by confining the assemblage between rigid impervious parallel sheets such as glass sheets in contact with the outer sheets of the assemblage. In a preferred embodiment, an additional porous carrier sheet saturated with hybridization buffer containing solvent capable of eluting RNA or DNA from the probe sheet but free from either probe or test bands of material is interposed between the outermost test sheet and the rigid glass plate in order to enhance diffusion of the labelled material through the stack of test sheets and provide a small reservoir of hybridization buffer.
Any conventional buffers can be employed for the transfer buffer or the hybridization buffer, but it is essential that the buffer or buffers initially saturating the probe sheet and the first test sheet immediately in contact with the probe sheet be free from solvent capable of eluting DNA or RNA from the probe sheet. All of the remaining test sheets must be saturated with a hybridization buffer which does contain solvent capable of eluting DNA or RNA from the probe sheet. There may be used as the eluting solvent such materials as formaldehyde or formamide, the latter being preferred. Neither the eluting solvent nor the buffers is capable of eluting DNA or RNA from the test sheets on which it is immobilized. While the precise amount of such eluting solvent in the buffer is not critical, it is desirable that it amount to 30 to 60% by weight of the total buffer. The remainder of the buffer, as well as the transfer buffer free from eluting solvent, may be of conventional composition; preferably the buffer contains sodium dodecyl sulfate (SDS) as a wetting agent. The length of time required for complete diffusion equilibration of the buffers to occur, which is desirable for best results, varies depending upon temperature and the total number of carrier sheets (as well as their thickness and porosity) in the stack or stacks. Although a temperature as low as about 30° C. can be used for hybridization in some cases, in order to ensure completion of the hybridization step it is preferred to heat the assemblage to a temperature of about 80°-100° C. and also to incubate it for a few hours at a temperature from room temperature to 42° C.
The present invention is particularly adapted for restriction mapping of a genome. In using it for this purpose, the sizes of the various DNA fragments generated by the desired number of different restriction enzymes is first determined, for example, by gel electrophoresis using known standards.
One of the digests is then selected for labelling, e.g. end-labelling with 32 P to serve as the probe. Preferably, the digest serving as a probe should contain a greater than average number of fragments and should contain no fragments that are so similar in size that they cannot be resolved by electrophoresis.
The desired map can be deduced by identifying and tabulating the particular fragments from each test sheet digest which hybridize with each fragment of the labelled probe sheet digest.
A kit for practicing the method of the invention includes a supply of porous carrier probe sheets and test sheets, a supply of buffer free from solvent capable of eluting DNA or RNA from a probe sheet, a supply of buffer containing a solvent capable of eluting DNA or RNA from a probe sheet, and optionally means for maintaining a probe sheet and at least one test sheet saturated with said buffers in static condition, and, if desired, a supply of labelled DNA or RNA fragments, and a supply of templates for cutting and pasting a restriction map. The means for maintaining the carrier sheets saturated with buffers under static condition, as described more fully in the Example which follows, may include tray, roller, glass or other impervious plates, and clamps.
The following specific example will serve as an illustration of the present invention without acting as a limitation upon its scope.
Several-microgram aliquots of adenovirus DNA were digested with the four restriction enzymes HindIII, EcoRl, BamHl and Kpnl. A small portion of one digest (HindIII) was end-labelled with 32 P nucleotides using the Klenow fragment of DNA polymerase I. (Alternatively, the fragments may be labelled using polynucleotide kinase or by allowing the T4 DNA polymerase to act first as a 3' 5' exonuclease in the absence of DNA precursors and then as a polymerase in the presence of 32 P dXTPs).
A few μg of DNA from each non-radioactive digest were then subjected to electrophoresis in a separate agarose gel (1% agarose in 40 mM Tris, 20 mM Na-Acetate, 1 mM EDTA, pH 7.5) by loading in a single 15 cm wide well. Electrophoresis was performed for a time compatible with having the fragments well separated with the smallest one no more than about 14 cm from the origin and at a low enough voltage (2 v/cm) so that the bands were sharp and undistorted across the width of the gel. The gels were stained with ethidium bromide and photographed with a ruler alongside to record the positions of the bands. Each gel was trimmed into a square 15 cm on a side with one corner (the bottom left) cut off at an angle for orientation purposes. (For the procedures that follow, the gel was never turned over so that orientation would not become confused.)
Each gel was then treated as for a Southern blot--denatured with 0.5M NaOH, 1M NaCl, neutralized with a buffer consisting of 1M Tris-HCl pH 7.5, 1M NaCl followed by 25 mM sodium phosphate, pH 6.5 and transferred to 15 cm. square nitrocellulose filter paper or preferably, to GeneScreen as a carrier sheet. The great advantages of "GeneScreen" are that it binds more quantitatively than nitrocellulose, especially the short DNA fragments, is much less subject to tearing and breaking, and does not permanently stick together in the sandwich. The orientation of the gel on the carrier sheet was marked on a corner of the sheet before transfer. Following the transfer, the carrier sheets were washed briefly in transfer buffer (25 mM sodium phosphate, pH 6.5) and vacuum-baked at 80° C. for 2-3 hr. to immobilize the unlabelled DNA bands on the sheet, forming test sheets.
The 32 P labelled DNA was subjected to similar electrophoresis using the 15 cm wide loading well. Again the gel was trimmed, a corner was cut off and the gel was prepared and blotted onto carrier sheet in the same manner as were the gels of the cold digests except that the baking step was omitted.
Several hours (or more, if desired) before the transfer of the labelled DNA was complete, one of the test sheets was soaked in degassed non-formamide hybridization buffer (5×SSC [SSC=0.15 M NaCl, 0.015 M Na-citrate], 25 mM sodium phosphate, pH 6.5, 10×Denhardt's solution [Denhardt's=0.02% Bovine serum albumin; 0.02% Ficoll 400; 0.02% polyvinyl pyrollidone], 0.1% SDS, 0.1% Na pyrophosphate, 200 μg/ml denatured non-homologous DNA) at 65° C. The other test sheets were soaked in degassed formamide hybridization buffer (5×SSC, 25 mM sodium phosphate, pH 6.5, 10×Denhardt's solution, 0.1% SDS, 0.1% Na pyrophosphate, 200 μg/ml denatured non-homologous DNA, 50% formamide).
The hybridization sandwich or stack could now be constructed. One clean, glass electrophoresis plate (20 cm×20 cm×4 mm) was placed flat on a sturdy surface such that the plate extended about 3 cm beyond the support on both the left and right sides. The probe sheet was removed from the transfer apparatus and rinsed in 25 mM sodium phosphate, pH 6.5 and placed on the gel plate. The surface of the paper adjacent to the gel during transfer was "face up" and the paper was oriented so that the high molecular weight DNA species were on the left side, and the low MW species on the right. Care was taken not to trap bubbles between the glass plate and the paper. A hard rubber artist's roller was used to roll the paper flat and expel air bubbles (rolling from front to back).
Next the test sheet in non-formamide hybridization buffer was laid face down exactly on top of the probe sheet but turned 90° with respect to the orientation of the probe sheet (with the large fragments toward the far edge), and air bubbles eliminated. Each band of DNA on the test sheet thus intersected each band on the probe sheet at right angles. The sheets were carefully rolled together with the artist's roller from front to back. Then one by one the other test sheets were laid down on top of the growing stack with their bands aligned with (parallel to) those of the first test sheet, being careful not to trap air bubbles between the sheets. When the stack was complete, the sheets were again rolled tightly together with the artist's roller from front to back. Most of the hybridization buffer was extruded from between the papers. Finally, a 15×15 cm piece of 0.33 MM thick cellulose chromatography paper (Whatman 3 MM paper) was soaked in the remaining formamide hybridization buffer and laid on top of the stack and rolled again. More formamide hybridization buffer containing formamide was poured on the top (3 MM) sheet, especially at the near edge. Then the edge of a second 20 cm×20 cm glass plate was placed along the near edge of the first plate and tilted down and away until the stack of sheets was sandwiched between the two glass plates. The plates were clamped together on the two sides. It was important that the pressure of the clamps be over the part of the plates under which the papers extend and that the sandwich be kept horizontal to minimize or eliminate further flow of the buffer.
Alternatively, a length of compressible rubber tubing can be placed between the plates around three sides of the stack of papers to act as a liquid seal, permitting the assemblage to be positioned vertically with the unsealed fourth side facing up to allow bubbles to escape during the subsequent heating step.
The hybridization cell or assemblage was incubated at room temperature one to two hours to allow the hybridization solutions to equilibrate, then placed in a 100°-110° C. oven until the hybridization buffer just began to boil or until a surface thermometer read 100°-110° C. Alternatively, heating to 80° C. is usually sufficient. Finally, the assemblage was incubated at 37°-42° C. overnight to complete the hybridization.
Following the hybridization, the glass plates were pried apart and the test sheets washed three times for 20 minutes each at 20° C. in 2×SSC, 0.1% SDS, then twice for 40 min. at 50° C. in 0.1×SSC, 0.1% SDS. The test sheets were dried and autoradiographed all in the same orientation (which was carefully noted). The 3 MM paper and the original 32 P labelled DNA probe sheet were placed in waterproof plastic envelopes and an autoradiogram of each was obtained to indicate the positions of the labelled DNA bands and the extent of transfer of labelled DNA through the stack of recipient blots and thus to serve as a guide in determining locations.
The information in the autoradiogram of the test sheets consists of spots of hybridization at the intersection of 32 P DNA bands with non-radioactive DNA bands that contain complementary sequences. The restriction map can be directly deduced from the positions of the hybridization spots. A labelled fragment is arbitrarily chosen as a starting place and the unlabelled fragments of a given digest that hybridize to that labelled fragment are placed adjacent to each other. The other labelled fragments that hybridize to these unlabelled fragments are then placed on the map appropriately next to the first labelled fragment. Thus one can "walk" in both directions down the DNA molecule, "stepping" first on a labelled fragment then on an unlabelled, then on a labelled, etc. Of course, if an unlabelled fragment is contained entirely within a labelled fragment, there will be only a single hybridization spot for that unlabelled fragment, but this does not interrupt the walk. The same procedure is then repeated, comparing each of the unlabelled digests to the labelled digest until all unlabelled digests have been mapped with respect to the labelled digest and thus to each other. Ambiguities in a single labelled-unlabelled comparison are almost always resolved by the time all unlabelled digests are mapped.
Labelled and/or unlabelled RNA can be substituted for the labelled and/or unlabelled DNA in the foregoing procedure without change except that in transferring the RNA to a porous sheet the steps of denaturing with NaOH and NaCl, neutralizing with Tris buffer, and with sodium phosphate buffer are omitted and the RNA is transferred directly, as is well known in the art.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US2783894 *||Mar 24, 1955||Mar 5, 1957||Millipore Filter Corp||Microporous nylon film|
|US3876738 *||Jul 18, 1973||Apr 8, 1975||Amf Inc||Process for producing microporous films and products|
|US4302204 *||Jul 2, 1979||Nov 24, 1981||The Board Of Trustees Of Leland Stanford Junior University||Transfer and detection of nucleic acids|
|US4455370 *||Mar 7, 1983||Jun 19, 1984||E. I. Du Pont De Nemours And Company||Transferring separated components in gel electrophoresis via nylon membrane|
|US4533628 *||Aug 3, 1983||Aug 6, 1985||New York University||Colony hybridization method|
|1||*||Masuda et al., (1979), Gene 6: 51 73.|
|2||Masuda et al., (1979), Gene 6: 51-73.|
|3||*||Ranki et al., (1983), Gene 21: 77 85.|
|4||Ranki et al., (1983), Gene 21: 77-85.|
|5||*||Sato et al., PNAS, vol. 74, 542 546, (1977).|
|6||Sato et al., PNAS, vol. 74, 542-546, (1977).|
|7||*||Southern, J. Mol. Biol., vol. 98, 503 517, (1975).|
|8||Southern, J. Mol. Biol., vol. 98, 503-517, (1975).|
|9||*||Wensink et al., Cell, vol. 18, 1231 46, (1979).|
|10||Wensink et al., Cell, vol. 18, 1231-46, (1979).|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US5202231 *||Jun 18, 1991||Apr 13, 1993||Drmanac Radoje T||Method of sequencing of genomes by hybridization of oligonucleotide probes|
|US5492806 *||Apr 12, 1993||Feb 20, 1996||Hyseq, Inc.||Method of determining an ordered sequence of subfragments of a nucleic acid fragment by hybridization of oligonucleotide probes|
|US5525464 *||Feb 28, 1994||Jun 11, 1996||Hyseq, Inc.||Method of sequencing by hybridization of oligonucleotide probes|
|US5667972 *||Jun 5, 1995||Sep 16, 1997||Hyseg, Inc.||Method of sequencing of genoms by hybridization of oligonucleotide probes|
|US5695940 *||Jun 5, 1995||Dec 9, 1997||Hyseq, Inc.||Method of sequencing by hybridization of oligonucleotide probes|
|US5776684 *||Dec 28, 1995||Jul 7, 1998||Edvotek||Method for staining biomolecules using a gelled matrix|
|US6018041 *||Jul 29, 1997||Jan 25, 2000||Hyseq, Inc.||Method of sequencing genomes by hybridization of oligonucleotide probes|
|US6025136 *||Aug 28, 1997||Feb 15, 2000||Hyseq, Inc.||Methods and apparatus for DNA sequencing and DNA identification|
|US6040193 *||Aug 4, 1998||Mar 21, 2000||Affymetrix, Inc.||Combinatorial strategies for polymer synthesis|
|US6054266 *||Nov 23, 1988||Apr 25, 2000||Applied Biosystems, Inc.||Nucleic acid detection with separation|
|US6107031 *||Feb 18, 1997||Aug 22, 2000||Hyseq, Inc.||Method for screening for unknown organisms|
|US6124102 *||Apr 21, 1998||Sep 26, 2000||Affymetrix, Inc.||Methods for determining receptor-ligand binding using probe arrays|
|US6136269 *||Apr 21, 1995||Oct 24, 2000||Affymetrix, Inc.||Combinatorial kit for polymer synthesis|
|US6197506||Apr 8, 1998||Mar 6, 2001||Affymetrix, Inc.||Method of detecting nucleic acids|
|US6225625||Jun 1, 1995||May 1, 2001||Affymetrix, Inc.||Signal detection methods and apparatus|
|US6261776||Apr 15, 1999||Jul 17, 2001||Affymetrix, Inc.||Nucleic acid arrays|
|US6291183||Nov 17, 1999||Sep 18, 2001||Affymetrix, Inc.||Very large scale immobilized polymer synthesis|
|US6291186||Mar 21, 2000||Sep 18, 2001||Lewis S. Gruber||Method for screening for unknown organisms|
|US6297006||Mar 4, 1997||Oct 2, 2001||Hyseq, Inc.||Methods for sequencing repetitive sequences and for determining the order of sequence subfragments|
|US6309822||Dec 23, 1996||Oct 30, 2001||Affymetrix, Inc.||Method for comparing copy number of nucleic acid sequences|
|US6309824||Jan 16, 1997||Oct 30, 2001||Hyseq, Inc.||Methods for analyzing a target nucleic acid using immobilized heterogeneous mixtures of oligonucleotide probes|
|US6310189||Jan 25, 2000||Oct 30, 2001||Affymetrix, Inc.||Nucleotides and analogs having photoremoveable protecting groups|
|US6329143||Aug 4, 1998||Dec 11, 2001||Affymetrix, Inc.||Very large scale immobilized polymer synthesis|
|US6344316||Jun 25, 1997||Feb 5, 2002||Affymetrix, Inc.||Nucleic acid analysis techniques|
|US6346413||Nov 17, 1999||Feb 12, 2002||Affymetrix, Inc.||Polymer arrays|
|US6355419||Apr 27, 1998||Mar 12, 2002||Hyseq, Inc.||Preparation of pools of nucleic acids based on representation in a sample|
|US6355432||Jun 2, 2000||Mar 12, 2002||Affymetrix Lnc.||Products for detecting nucleic acids|
|US6379895||Sep 1, 2000||Apr 30, 2002||Affymetrix, Inc.||Photolithographic and other means for manufacturing arrays|
|US6383742||Aug 15, 1997||May 7, 2002||Radoje T. Drmanac||Three dimensional arrays for detection or quantification of nucleic acid species|
|US6395491||Jun 2, 2000||May 28, 2002||Affymetrix, Inc.||Method of information storage and recovery|
|US6403320||Oct 5, 2000||Jun 11, 2002||Affymetrix, Inc.||Support bound probes and methods of analysis using the same|
|US6403957||Oct 16, 2000||Jun 11, 2002||Affymetrix, Inc.||Nucleic acid reading and analysis system|
|US6406844||Jun 1, 1995||Jun 18, 2002||Affymetrix, Inc.||Very large scale immobilized polymer synthesis|
|US6410229||Dec 14, 1998||Jun 25, 2002||Affymetrix, Inc.||Expression monitoring by hybridization to high density nucleic acid arrays|
|US6416952||Sep 1, 2000||Jul 9, 2002||Affymetrix, Inc.||Photolithographic and other means for manufacturing arrays|
|US6420169||Nov 30, 1994||Jul 16, 2002||Affymetrix, Inc.||Apparatus for forming polynucleotides or polypeptides|
|US6440667||Jul 28, 1999||Aug 27, 2002||Affymetrix Inc.||Analysis of target molecules using an encoding system|
|US6451536||Sep 27, 2000||Sep 17, 2002||Affymetrix Inc.||Products for detecting nucleic acids|
|US6451996||Jan 21, 2000||Sep 17, 2002||Callida Genomics, Inc.||Method of sequencing of genomes by hybridization of oligonucleotide probes|
|US6468740||Apr 8, 1998||Oct 22, 2002||Affymetrix, Inc.||Cyclic and substituted immobilized molecular synthesis|
|US6491871||Dec 9, 1997||Dec 10, 2002||Affymetrix, Inc.||System for determining receptor-ligand binding affinity|
|US6506558||Nov 29, 1995||Jan 14, 2003||Affymetrix Inc.||Very large scale immobilized polymer synthesis|
|US6544739||Jun 2, 2000||Apr 8, 2003||Affymetrix, Inc.||Method for marking samples|
|US6545264||Aug 26, 1999||Apr 8, 2003||Affymetrix, Inc.||Systems and methods for high performance scanning|
|US6548257||Aug 22, 2001||Apr 15, 2003||Affymetrix, Inc.||Methods of identifying nucleic acid probes to quantify the expression of a target nucleic acid|
|US6551784||May 9, 2001||Apr 22, 2003||Affymetrix Inc||Method of comparing nucleic acid sequences|
|US6566495||Dec 17, 1999||May 20, 2003||Affymetrix, Inc.||Very large scale immobilized polymer synthesis|
|US6576424||Jan 25, 2001||Jun 10, 2003||Affymetrix Inc.||Arrays and methods for detecting nucleic acids|
|US6600031||Apr 21, 1998||Jul 29, 2003||Affymetrix, Inc.||Methods of making nucleic acid or oligonucleotide arrays|
|US6610482||Apr 24, 2000||Aug 26, 2003||Affymetrix, Inc.||Support bound probes and methods of analysis using the same|
|US6630308||Dec 6, 2001||Oct 7, 2003||Affymetrix, Inc.||Methods of synthesizing a plurality of different polymers on a surface of a substrate|
|US6646243||Mar 15, 2002||Nov 11, 2003||Affymetrix, Inc.||Nucleic acid reading and analysis system|
|US6660234||May 15, 2002||Dec 9, 2003||Affymetrix, Inc.||Apparatus for polymer synthesis|
|US6747143||May 20, 2002||Jun 8, 2004||Affymetrix, Inc.||Methods for polymer synthesis|
|US6849462||May 26, 2000||Feb 1, 2005||Affymetrix, Inc.||Combinatorial strategies for polymer synthesis|
|US6864101||May 26, 2000||Mar 8, 2005||Affymetrix, Inc.||Combinatorial strategies for polymer synthesis|
|US6919211||Sep 1, 2000||Jul 19, 2005||Affymetrix, Inc.||Polypeptide arrays|
|US6927032||Jan 28, 2003||Aug 9, 2005||Affymetrix, Inc.||Expression monitoring by hybridization to high density oligonucleotide arrays|
|US6943034||Feb 4, 2000||Sep 13, 2005||Affymetrix, Inc.||Combinatorial strategies for polymer synthesis|
|US6955915||Dec 14, 2001||Oct 18, 2005||Affymetrix, Inc.||Apparatus comprising polymers|
|US7087732||Dec 28, 2001||Aug 8, 2006||Affymetrix, Inc.||Nucleotides and analogs having photoremovable protecting groups|
|US7323298||Jul 30, 1996||Jan 29, 2008||The Board Of Trustees Of The Leland Stanford Junior University||Microarray for determining the relative abundances of polynuceotide sequences|
|US7378236||Aug 14, 1995||May 27, 2008||The Board Of Trustees Of The Leland Stanford Junior University||Method for analyzing gene expression patterns|
|US7442499||Nov 24, 1998||Oct 28, 2008||The Board Of Trustees Of The Leland Stanford Junior University||Substrates comprising polynucleotide microarrays|
|US7691330||May 26, 2000||Apr 6, 2010||Affymetrix, Inc.||Combinatorial strategies for polymer synthesis|
|US7736906||Jun 17, 2002||Jun 15, 2010||Affymetrix, Inc.||Combinatorial strategies for polymer synthesis|
|US8236493||Aug 7, 2008||Aug 7, 2012||Affymetrix, Inc.||Methods of enzymatic discrimination enhancement and surface-bound double-stranded DNA|
|US20050158746 *||Nov 23, 2004||Jul 21, 2005||Affymetrix Inc.||Expression monitoring by hybridization to high density oligonucleotide arrays|
|US20050158772 *||Dec 23, 2004||Jul 21, 2005||Affymetrix, Inc.||Nucleic acid analysis techniques|
|US20050202500 *||Apr 28, 2005||Sep 15, 2005||Affymetrix, Inc.||Expression monitoring to high density oligonucleotide arrays|
|WO1990004652A1 *||Oct 23, 1989||May 3, 1990||Dnax Research Inst Of Molecula||Dna sequencing by multiple mixed oligonucleotide probes|
|U.S. Classification||435/6.19, 436/501, 536/24.3, 436/808, 436/504, 435/810|
|Cooperative Classification||Y10S436/808, Y10S435/81, C12Q1/6834, C12Q1/6813|
|European Classification||C12Q1/68B10, C12Q1/68B|
|Jan 23, 1984||AS||Assignment|
Owner name: PRESIDENT AND FELLOWS OF HARVARD COLLEGE, CAMBRIDG
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:POTTER, HUNTINGTON;REEL/FRAME:004223/0430
Effective date: 19840116
|Mar 2, 1990||FPAY||Fee payment|
Year of fee payment: 4
|Mar 3, 1994||FPAY||Fee payment|
Year of fee payment: 8
|Apr 14, 1998||REMI||Maintenance fee reminder mailed|
|Sep 20, 1998||LAPS||Lapse for failure to pay maintenance fees|
|Dec 1, 1998||FP||Expired due to failure to pay maintenance fee|
Effective date: 19980923